Erynia neoaphidis is the most widespread aphid pathogen in temperate regions. A few authors have identified this species as infecting some non-aphid hosts. It exhibits high variability with respect to morphology and cultural characteristics that underlines the need for assessment of the genetic variability within this species. We used size of PCR-amplified ribosomal DNA internal transcribed spacer (ITS) coupled with RAPD-PCR pattern in order to investigate genetic variability within a sample of 30 isolates identified as E. neoaphidis and one isolate of E. kondoiensis from diverse countries and hosts. Isolates fell into four discrete ITS size groups: 575 base pairs (bp) in isolates from non-aphid hosts, 1000 (including the E. kondoiensis isolate), 1100, or 1450 bp in those from Aphididae. The 1100-type seems to be the one of most widespread, since two-thirds of the isolates from diverse locations belong to this type. ITS types can be correlated with morphological and physiological characteristics. A great deal of genetic variability exists within ITS types, as shown by RAPD-PCR. RAPD groupings could be related with geographical origin in some cases. There was no apparent relationship between host and ITS type or RAPD pattern among isolates from Aphididae. PCR-based technologies provide evidence that could be used to clarify the taxonomy of E. neoaphidis and closely related species, and will help identify markers related to epidemiological characteristics.